Pathogenic Yersiniae are excellent models for the study of bacterial-host interactions at the molecular level. Important paradigms -- such as contact-dependent Type III secretion systems that deliver bacterial proteins to the cytoplasm of host cells -- have emerged from the study of three pathogenic species: Y. enterocolitica and Y. pseudotuberculosis, which are enteropathogens causing a wide range of gastrointestinal symptoms; and Y. pestis, which is the etiological agent of plague. Type III secretion systems (TTSS) were first described in Yersiniae, and the well-characterized Ysc TTSS is found in all three pathogenic species. Our long-term goal is to understand the virulence factors that distinguish high-virulent from low-virulent strains of Y. enterocolitica. One such virulence factor is a second contact-dependent TTSS encoded by genes of the YSA locus, a putative pathogenicity island. This TTSS remains particularly understudied and is the focus of this proposal. The Ysa TTSS secretes a set of proteins called Ysp's and functions independently from the plasmid-encoded Ysc TTSS. Studies with both animal and cellular models of infection indicate that the Ysa TTSS is important for pathogenesis. At the cellular level macrophages are thought to play a significant role in limiting Y. enterocolitica infection. Notably, we have shown that cultured macrophages respond differently to infection by Ysa TTSS mutants compared to wild type Y. enterocolitica. The proposed research will extend these results to understand how the Ysa TTSS contributes to pathogenesis at the molecular level, and in cellular and animal models. We hypothesize that the Ysa TTSS targets one or more virulence effectors into host cells and that these in turn influence disease progression. To address this hypothesis, we have initiated three specific aims: (1) Identify virulence effectors targeted to the host cell by the Ysa TTSS; (2) Define the regulatory network that controls Ysa TTSS gene expression; and (3) Determine the role of the secreted Ysps and the Ysa TTSS in pathogenesis using animal and cell culture models of infection. This research will contribute to understanding why some pathogens have multiple TTSS systems. More specifically, it will address the question of why high-virulent Y. enterocolitica maintain two contact-dependent TTSS systems, each of which transports a defined set of proteins and each of which is under separate control.